LC-MS/MS standardization and validation of glycyrrhizin from the roots of Taverniera cuneifolia: A potential alternative source of Glycyrrhiza glabra

Glycyrrhizin is a triterpene glycoside derived from Glycyrrhiza glabra and related species which is a renowned phytochemical used to cure a variety of ailments such as inflammation, sore throat, hepatitis etc. It is in huge demand owing to its various valuable properties. With the ever-increasing demand of glycyrrhizin, the search for alternative sources for glycyrrhizin is on rise. One such species with a scientific basis and good concentration of glycyrrhizin is Taverniera cuneifolia. A thin-layer chromatography (TLC) method was established to determine the presence of glycyrrhizin in T. cuneifolia. Further, standardisation and validation, a High performance liquid chromatography (model NEXERA-X2) with LCMS system (Model LCMS-8040) from Shimadzu were used. The analysis was performed by using shim-pack XR-ODS, C18 (75 mm × 3.0 mm) 2.2 μm. In this analysis, the mobile phase used was a combination of acetonitrile and a 20 mM ammonium acetate buffer that was subjected to gradient time programming and monitored by Multiple Reaction Monitoring (MRM) in positive ion mode. The method was validated for linearity, accuracy, precision, recovery, detection, and quantitation limit. The technique was confirmed to be linear within the concentration range of 5 ng/mL to 500 ng/mL with R2 > 0.991. The LOD and the LOQ were 2 ng/mL and 5 ng/mL respectively. The suggested approach satisfied the acceptance criteria for linearity, accuracy, precision, specificity, robustness, LOD, LOQ, and system adaptability.


Introduction
Glycyrrhizin is a triterpene glycoside derived from the licorice root (Glycyrrhiza glabra). It contains glycyrrhetinic acid and two molecules of glucuronic acid at the C-3 position. Glycyrrhizin has been confirmed to be effective in the treatment of several types of liver inflammation [1,2,3,4,5,6,7,8,9,10,11], lung [12], kidney, intestine, and spinal cord [13]. Currently, It has been proven to be effective in considerably lowering steatosis and necrosis of liver cells [14], inhibition of lung cancer and fibro sarcomas [12], treatment of Hepatitis C [15], potent inhibitor of bile acid-induced apoptosis and necrosis [16]. Glycyrrhizin also exhibits proapoptotic properties in a hepatocyte model of cholestatic liver injury [16], antiviral activity and chemo-preventive activity [17,18], Suppresses SARS-CoV multiplication, as well as virus adsorption and penetration, at an early stage of the replicative cycle [19,20].
Glycyrrhizin is one of the promising phyto-molecule in present day scenario. With ever increasing demand of glycyrrhizin, the search for alternative sources has becomes essential to fulfil the gap of demand and supply. One such species with the presence of glycyrrhizin and other similar phyto-constitiuents (vanillic, syringic, ferulic, o-coumaric, melilotic, and p-Hydroxy benzoic acids and sugars [21]) with that of liquorice is Taverniera cuneifolia [22,23,24]. However, glycyrrhizin content in the species is yet to standardized and validated. Taverniera cuneifolia (TC) belonging to the family of Fabaceae is endemic to Northeast African and Southwest Asian countries. It's been used as an expectorant, blood purifier, anti-inflammatory, wound healer, antiulcer, and used to treat spleen tumours [25] for generations. With the above perspectives, attempts have been made to detect, quantify and validate the presence of glycyrrhizin in Taverniera cuneifolia.

Reagents, standards, and solutions
Solvents (Butanol, water, acetonitrile, methanol) and chemicals (acetic acid and ammonium acetate) were of analytical grade or HPLC grade procured from Merck specialties India Pvt. Ltd., India. Glycyrrhizic acid mono-ammonium salt (purity 95 %) were brought from Sigma Aldrich chemie (steinheim Germany). Deionized water was obtained by using Milli-Q syste.

Collection of samples
The roots of T. cuneifolia were collected in the month of July from Munjakagam, Saurashtra University, Rajkot, Gujarat, India. Roots were properly cleaned under the running water for 10-15 min and then shade dried for a period of 4 days. The dried roots were pulverised in a mixer grinder and stored in a zip lock plastic bag at room temperature.

Thin layer chromatography analysis
Finely powdered plant material was extracted with 10 mL of methanol by sonication for 15 min. The extract was taken and applied band-wise onto a silica gel 60 F254 TLC sheet (Merck); First and second bands served as reference standards. The mobile phase was a mixture of butanol: acetic acid: water (6:1:3 v/v) [22]. The plates were developed, dried, and treated with anisaldehyde sulphuric acid reagent before being used. The plate was examined under UV light after being heated.
2.4. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis 2.4.1. Preparation of LC samples 1 g of dried powder was diluted and sonicated for 15 min in 5 mL of 1:1 Acetonitrile: Water, which was centrifuged at 5000 RPM (at room temperature) for 10 min and the resultant supernatant of TC and GG was diluted to the concentration of 0.1 mg mL À1 and 0.01 mg mL À1 respectively for LC-MS/MS analysis.

Liquid chromatography-mass spectrometry condition
Chromatographic development was performed on Shimadzu NEXERA-X2, UHPLC (Ultra High Performance Liquid Chromatograph) system with LC-30AD pumps, SIL-30A autosampler and CTO-20AC as column oven. The data processor used LabSolutions software. Analysis was performed using APCI (Atmospheric Pressure Chemical Ionization) interface at positive mode with a capillary voltage of 4 V. The following MS parameters were used in the analysis: Nebulizing Gas flow: 2 L min À1 , Drying Gas flow: 15 L min À1 , Interface temperature: 350 C, DL (desolvation Line) temperature: 200 C, and Heating Block: 400 C.

Method validation
The suggested analytical method has been validated to confirm that it was suitable for its actual purpose. The method has been validated as per the ICH guidelines for specificity, linearity, range, accuracy, precision, sensitivity (LOQ and LOD) as validation parameters.

Linearity
A stock solution with 10,000 ng/mL concentration of glycyrrhizin standard was prepared in water and acetonitrile. Different aliquots were made to acquire six different desired concentrations ranging from 5 ng/mL to 500 ng/mL which were injected (5 μL each) by autosampler and chromatographed according to the previously mentioned protocol. To avoid degradation due to light exposure, the stock solution was stored in the dark. The experiment was performed in triplicate,  and the average was taken in the calculations. Peak area was plotted against analyte concentrations to generate the calibration graph and data were statistically analysed using correlation and least square linear regression.

Specificity
The specificity of the method was determined by comparing the standard and sample. The peak for Glycyrrhizin was validated by comparing the sample's retention time and spectra to that of the standard. The assay developed through HPLC method was employed for Glycyrrhizin determination in methanolic extract of Taverniera cuneifolia and Glycyrrhiza glabra root. For this the sample working solution of 5 μL was injected and the area of these two were measured and quantitated against calibration curve.

Accuracy as recovery
Standard was introduced into the pre-analyzed samples at three different concentration levels. i.e., 50 ng/mL, 100 ng/mL and 200 ng/mL and the mixtures were then re-analyzed with the proposed technique. The recovery experiment was repeated thrice, and the percentage recovery of the result was calculated. The method was performed in triplicate (Table 2) and the representative chromatograms of GG and TC are shown in Figure 5. With the above-mentioned solvent system, the GG root extract was diluted 20,000 times, whereas the TC root extract was diluted 2000 times.

Precision
Experiments involving repeatability and intermediate precision were carried out to determine the precision of the method. Three different concentrations of standard were prepared and applied on the same day as well as three separate days to determine inter-and intra-day precisions. Assay for each analysis was calculated and % RSD was determined.

Limit of detection (LOD) and limit of quantification (LOQ)
The method's sensitivity was calculated using measurements of the limit of detection (LOD) and limit of quantification (LOQ). The concentration of a sample resulting in a signal-to-noise ratio of three was designated as the LOD, while the concentration of a sample resulting in a signal-to-noise ratio of ten was assigned as the LOQ.

TLC of T. cuneifolia and G. glabra
TLC for glycyrrhizin in T. cuneifolia roots was carried out in conjunction with G. glabra during the present study. Several solvent systems, including butanol, glacial acetic acid, and water [22,26], were tried during the method development phase of this study. The best result was obtained using an optimised combination of butanol, glacial acetic acid, and water (6:1:3 v/v). The brown colour band was clearly apparent in the extracts at Rf 0.5 (Figure 1).

LC-MS/MS summary
Satisfactory separation was obtained for ionizing the glycyrrhizin in the mass spectrometer using the APCI (þ) ionization mode with corona discharge voltage of 4 V. The desolvation temperature was kept low at 200 C to prevent glycyrrhizin from thermal decomposition.

Linearity
A good linearity was achieved in the concentration ranges of 5 ng/ mL-500 ng/mL Glycyrrhizin (Figures 3 and 4). The correlation of coefficient was R 2 ¼ 0.9997 (Table 1).

Specificity
The retention time of Taverniera cuneifolia and Glycyrrhiza glabra root extracts was 2.753 and 2.734 respectively ( Figure 5).
The mean assay value of TC at 150 ppm with % RSD was 0.83 whereas the mean assay value of GG at 850 ppm with % RSD was 0.77. As a result, it was found that the procedure was more selective and specific.

Accuracy as recovery
Three replicates of glycyrrhizin were taken in the range of 50 ng/mL, 100 ng/mL and 200 ng/mL for recovery determination and the mean recovery obtained was 89 %.

Precision
The percent RSD for intra-day and inter-day precision for Glycyrrhizin were 0.81 and 0.48 percent, respectively (Table 1).

Limit of detection (LOD) and limits of quantitation (LOQ)
The LOD and LOQ of Glycyrrhizin were discovered to be 2 ng/mL and 5.0 ng/mL respectively (Table 1).

Glycyrrhizin quantification in plant extracts using LC-MS/MS
The proposed method was utilised to assess the concentration of glycyrrhizin in plant root extracts from TC and GG. It was found that the content of glycyrrhizin was 8,681,997.68 ng/mL in GG root extract and 153,072.85 ng/mL in TC root extract respectively ( Table 2). This will be the first report of a validated technique for rapid detection and quantification of glycyrrhizin in TC root extract versus GG.

Discussion
Licorice (Glycyrrhiza glabra), also known as Mulethi or Yastimadhu, is a well-known medicinal plant around the world for its flavoring and therapeutic benefits and is employed in about 1250 herbal formulas. Ethnobotanical and Ayurvedic research states that T. cuneifolia (Jalaj Yashtimadu) is utilized as an alternative for G. glabra (Sthalaj Yashtimadu). Consequently, to prove whether T. cuneifolia could be used as an alternative of standard drug Glycyrrhiza glabra, a protocol was developed to estimate glycyrrhizin in T. cuneifolia. In comparison to G. glabra, the new approach reveals that Taverniera cuneifolia has a substantial amount of glycyrrhizin. The significance of this study lies in the fact that, for the first time, the measurement of glycyrrhizin in T. cuneifolia relative to G. glabra was standardized and confirmed using LC-MS/MS, while the data available to date are limited to HPTLC analysis with no quantitative calculation. In order to standardize this technique, ammonium acetate buffer (20 mM) was used for better resolution and absolute peak of Glycyrrhizin. The validation of Glycyrrhizin in T. cuneifolia opines a new avenue in various pharmaceutical industry as an anti-inflammatory, antiallergic, anti-cancer agent [15,22,23,24,27] and as a herbal supplement for cough syrups, chewing gums, beverages, and candies. It even opens the feasibility of using T. cuneifolia as and an alternative to 1250 herbal formulations made by G. glabra. Thus, Taverniera cuneifolia is a prospective plant for the present and future, with a wide range of industrial uses.

Conclusion
The development and validation of a sensitive and efficient LC-MS/ MS approach for identifying the glycyrrhizin content in Taverniera cuneifolia has been established.
The method has been validated for investigating active principles in the complex mixture of herbal ingredients. The method could be extended to include marker-based standardisation of other herbal products containing Glycyrrhizin.
The technique was found to be easy, sensitive, precise, accurate, specific and it could be employed for routine quality monitoring of herbal raw materials as well as the quantification of Glycyrrhizin in plant materials. The validated LC-MS/MS process is useful for routine quality control analysis.

Author contribution statement
Mannu Dwivedi: Preliminary Analysis and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Shailendra Rane: Performed the experiments; Contributed reagents, materials, analysis tools or data.
Padamnabhi S. Nagar: Conceived and designed the experiments.

Funding statement
This work was supported by the National Medicinal Plant Board, AYUSH, Government of India.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper.